猪瘟病毒疫苗株反向遗传学改造
发布时间：2019-03-25 07:59
【摘要】：猪瘟(Classical swine fever,CSF),别名猪霍乱,俗称“烂肠瘟”,是猪的一种高感染性的疾病,致死率高达90%以上,是威胁养猪业的主要传染病之一。猪瘟的爆发会导致严重的经济损失,被世界动物卫生组织列为A类传染病,受到了各个国家的高度重视,成为了各个国家研究的热点。猪瘟的病原猪瘟病毒是一种包膜病毒,大小约40-60nm,它的基因组大小约12.3kb,是单股正链RNA,包含了一个大的开放阅读框(ORF),一个5'非编码区(5'UTR)和一个3'非编码区(3'UTR)。ORF能够编码大约3900个氨基酸的多聚蛋白,然后在信号肽酶等作用下产生12个成熟蛋白,顺序依次为Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B。其中,C、Erns、E1、E2为4个结构蛋白,Npro、P7、NS2、NS3、NS4A、NA4B、NS5A、NS5B为8个非结构蛋白。本研究第一部分根据实验室已构建的猪瘟病毒感染性的c DNA,通过插入CMV启动子和锤头状核酶构建了重组质粒p MC18-CMV-HM-CSFV,并用于猪瘟病毒的拯救。为了验证锤头状核酶的功能,我们构建了p GL2.0-CMV、p GL2.0-CMV-HM、p GL2.0-C MV-HM'和p GL2.0-CMV-HM-5'UTR四个质粒。通过双荧光素酶报告基因检测系统和实时荧光定量核酸扩增检测系统(Real-time Quantitative PCR Detecting Syste m,QPCR)检测发现,重组质粒转染进细胞后,锤头状核酶能够发挥约62%的剪切作用。经过QPCR检测后发现,改造后的质粒p MC18-CMV-HM-CSFV通过转染进细胞成功拯救出了病毒。本研究为猪瘟疫苗的发展奠定了基础。本研究第二部分探究了猪瘟病毒C蛋白与5'UTR的关系。在实验室的前期研究中发现,猪瘟病毒的C蛋白对3'UTR具有调节作用。为了确定C蛋白与5'UTR之间是否存在相互作用关系,我们分别构建了双荧光素酶表达质粒p MIR-5'UTR-RLUC和p MIRRLUC。通过双荧光素酶报告基因检测系统检测证明,p MIR-5'UTR-RLUC转染进细胞后,5'UTR能够发挥相应的作用。将猪瘟病毒C蛋白的真核表达载体p CMV-C与5’UTR的真核表达载体p MIR-5'UTR-RLUC共转染至PK-15细胞,然后通过western blotting检测、实时荧光定量核酸扩增检测系统(Real-time Quantitative PCR Detecting System)和双荧光素酶报告基因检测系统(Dual-Luciferase Reporter Assay System)证明,C蛋白与5'UTR之间不存在相互作用关系。本部分研究为探究猪瘟病毒蛋白对5'UTR功能的影响奠定了基础。
[Abstract]:Swine cholera (Classical swine fever,CSF), also known as swine cholera, is a highly infectious disease of pigs with a mortality rate of more than 90%. It is one of the main infectious diseases that threaten the pig industry. The outbreak of classical swine fever will lead to serious economic losses. It has been classified as a class A infectious disease by the World Organization for Animal Health (OIE). It has been attached great importance to by various countries and has become the focus of research in various countries. The pathogen of classical swine fever (CSFV) is a envelope virus with a genome size of about 12.3kb and a large open reading frame (ORF) (ORF), contained in the single strand positive strand RNA, which is about 40 脳 60 nm in size and has a genome size of about 12.3 kb.Swine fever virus is the pathogen of classical swine fever. A 5 'non-coding region (5'UTR) and a 3' non-coding region (3'UTR). ORF encodes a Polymeric protein of about 3900 amino acids, and then produces 12 mature proteins under the action of signal peptidase, etc. Order is Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Among them, C, Erns, E1, E2 and Npro,P7,NS2,NS3,NS4A,NA4B,NS5A,NS5B are four structural proteins and eight non-structural proteins. In the first part of this study, the recombinant plasmid p-DNA, was constructed by inserting CMV promoter and hammerhead ribozyme into the infectious c-MC18-CMV-HM-CSFV, of classical swine fever virus (CSFV) which had been constructed in the laboratory and used for the rescue of CSFV. To verify the function of hammerhead ribozyme, four plasmids, p GL2.0-CMV,p GL2.0-CMV-HM,p GL2.0-CMV-HM 'and p GL2.0-CMV-HM-5'UTR, were constructed. Double luciferase reporter gene detection system and real-time fluorescence quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting Syste-m, QPCR) showed that the hammerhead ribozyme could perform about 62% of the cleavage effect after the recombinant plasmid was transfected into the cells. After QPCR detection, it was found that the modified plasmid p-MC18-CMV-HM-CSFV successfully saved the virus by transfection into the cells. This study laid a foundation for the development of swine fever vaccine. In the second part of this study, the relationship between C protein and 5'UTR of classical swine fever virus (CSFV) was investigated. In previous laboratory studies, it was found that the C protein of Classical Swine Fever virus (CSFV) has a regulatory effect on 3'UTR. In order to determine the interaction between C protein and 5'UTR, we constructed double luciferase expression plasmids p MIR-5'UTR-RLUC and p MIRRLUC., respectively. The double luciferase reporter gene detection system showed that 5'UTR could play a role in the transfection of p-MIR-5'UTR-RLUC into cells. The eukaryotic expression vector p-CMV-C of CSFV C protein was co-transfected with the eukaryotic expression vector p-MIR-5'UTR-RLUC of 5'UTR into PK-15 cells, and then detected by western blotting. Real-time fluorescent quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting System) and double luciferase reporter gene detection system (Dual-Luciferase Reporter Assay System) showed that there was no interaction between C protein and 5'UTR. This part of the study laid a foundation for exploring the effect of classical swine fever virus protein on the function of 5'UTR.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
本文编号：2446793
[Abstract]:Swine cholera (Classical swine fever,CSF), also known as swine cholera, is a highly infectious disease of pigs with a mortality rate of more than 90%. It is one of the main infectious diseases that threaten the pig industry. The outbreak of classical swine fever will lead to serious economic losses. It has been classified as a class A infectious disease by the World Organization for Animal Health (OIE). It has been attached great importance to by various countries and has become the focus of research in various countries. The pathogen of classical swine fever (CSFV) is a envelope virus with a genome size of about 12.3kb and a large open reading frame (ORF) (ORF), contained in the single strand positive strand RNA, which is about 40 脳 60 nm in size and has a genome size of about 12.3 kb.Swine fever virus is the pathogen of classical swine fever. A 5 'non-coding region (5'UTR) and a 3' non-coding region (3'UTR). ORF encodes a Polymeric protein of about 3900 amino acids, and then produces 12 mature proteins under the action of signal peptidase, etc. Order is Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Among them, C, Erns, E1, E2 and Npro,P7,NS2,NS3,NS4A,NA4B,NS5A,NS5B are four structural proteins and eight non-structural proteins. In the first part of this study, the recombinant plasmid p-DNA, was constructed by inserting CMV promoter and hammerhead ribozyme into the infectious c-MC18-CMV-HM-CSFV, of classical swine fever virus (CSFV) which had been constructed in the laboratory and used for the rescue of CSFV. To verify the function of hammerhead ribozyme, four plasmids, p GL2.0-CMV,p GL2.0-CMV-HM,p GL2.0-CMV-HM 'and p GL2.0-CMV-HM-5'UTR, were constructed. Double luciferase reporter gene detection system and real-time fluorescence quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting Syste-m, QPCR) showed that the hammerhead ribozyme could perform about 62% of the cleavage effect after the recombinant plasmid was transfected into the cells. After QPCR detection, it was found that the modified plasmid p-MC18-CMV-HM-CSFV successfully saved the virus by transfection into the cells. This study laid a foundation for the development of swine fever vaccine. In the second part of this study, the relationship between C protein and 5'UTR of classical swine fever virus (CSFV) was investigated. In previous laboratory studies, it was found that the C protein of Classical Swine Fever virus (CSFV) has a regulatory effect on 3'UTR. In order to determine the interaction between C protein and 5'UTR, we constructed double luciferase expression plasmids p MIR-5'UTR-RLUC and p MIRRLUC., respectively. The double luciferase reporter gene detection system showed that 5'UTR could play a role in the transfection of p-MIR-5'UTR-RLUC into cells. The eukaryotic expression vector p-CMV-C of CSFV C protein was co-transfected with the eukaryotic expression vector p-MIR-5'UTR-RLUC of 5'UTR into PK-15 cells, and then detected by western blotting. Real-time fluorescent quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting System) and double luciferase reporter gene detection system (Dual-Luciferase Reporter Assay System) showed that there was no interaction between C protein and 5'UTR. This part of the study laid a foundation for exploring the effect of classical swine fever virus protein on the function of 5'UTR.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
【参考文献】
相关期刊论文 前1条
1 侯玉臻;赵丹彤;刘国英;贺番;刘斌;付绍印;郝永清;张文广;;猪瘟病毒核心蛋白研究进展[J];病毒学报;2015年05期
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